| Objective:Crucian carp(Carassius auratus)is an important freshwater farmed fish in China.It is highly favored by consumers because of its tender meat and delicious taste.However,in recent years,the gill hemorrhage caused by Cyprinid herpesvirus 2(CyHV-2)has brought huge economic losses to the crucian carp breeding industry.CyHV-2 is a double-stranded DNA virus in the family Herpesviridae.This virus has a very high lethality rate to fish,such as goldfish,crucian carp,and allogynogenetic crucian carp.So far,there is still a lack of effective prevention and treatment methods for CyHV-2 infection.Relevant literatures showed that yolk antibody could reduce the infection of Vibrio,Aeromonas hydrophila and Vibrio parahaemolyticus,and has made good progress in the treatment of vibriosis,sepsis and acute hepatopancreas necrosis.In this study,we use the recombinant capsid and envelope proteins of CyHV-2 as the immunogen to prepare a specific yolk antibody,and verified the function of the yolk antibody by in vivo and in vitro experiments.In addition,we preliminarily explored the protective mechanism of yolk antibody,aiming to provide a new treatment for gill hemorrhage caused by CyHV-2.Methods:(1)An Escherichia coli prokaryotic expression system was developed by constructing recombinant pET28a-D4ORFs expression vector.Recombinant D4ORFs protein was generated and verified by using Western blot analysis.(2)The purified D4ORFs recombinant protein was used as immunogen to prepare anti-CyHV-2 egg yolk IgY(anti-CyHV-2 IgY).The titer and the specificity of the egg yolk IgY were detected by using ELISA assay and Western blot analysis.The yolk antibody powder was prepared by freeze-drying and spray-drying techniques,and the ELISA assay was used to evaluate the titer of anti-CyHV-2 IgY and thus to optimize the optimal method for processing of the yolk antibody.(3)The stock anti-CyHV-2 IgY solution(1 g/mL)was diluted by M199 cell culture medium with a ratio of 1:100 and 1:200,respectively.The diluted IgY solutions were mixed with CyHV-2 virus solution,and used to incubate crucian carp brain cells(GiCB)at 25℃ for 4-14 d in antibody neutralization experiments.The cytopathic effect(CPE)of the anti-CyHV-2 IgY was observed.We prepared functional feed by including 0.1%,0.3%,and 0.6%of the anti-CyHV-2 IgY to feed allogynogenetic crucian carp at water temperature of 16 ± 2℃ for 25 days.Then,the experimental fish were immersed in CyHV-2 solution(1.8 ×105 copies/mL)for 24 hours and were observed for 21 days.The relative immune protection rate of each experimental group was statistically analyzed.The transmission electron microscope was used to observe the morphological characteristics of CyHV-2 virus particles.The eosin-hematoxylin staining(HE)was used to observe tissue structure damage.The real-time fluorescence quantitative PCR(qPCR)was used to detect the viral load of CyHV-2,so as to screen out the optimal dosage of anti-CyHV-2 IgY for the best preventive effect.(4)Under a water temperature at 16 ± 2℃,the allogynogenetic crucian carp was immersed in CyHV-2 solution(1.8 ×105 copies/mL)for 24 h.The fish were then fed for 21 days with the functional feed that showed the best preventive effect.The relative immune protection rate in each experimental group was analyzed.The trunk kidney of the moribund allogynogenetic crucian carp was extracted,and the viral load and the relative expression of CyHV-2 immune-related genes were detected by qPCR.We extracted the trunk kidney and spleen to examine the pathological damage by HE staining.After 21 days of infection,the blood of the survived fish was taken for the detection of serum biochemical parameters and blood routine indicators to evaluate the therapeutic effect of anti-CyHV-2 egg yolk IgY.Results:We constructed the recombinant plasmid pET28a-D4ORFs,and induced the recombinant D4ORFs protein.We prepared specific anti-CyHV-2 IgY,and worked out freeze-drying as the best processing method,The in vitro virus neutralization experiments showed that,when infected with CyHV-2 virus for 9 days,GiCB cells in the anti-CyHV-2 IgY group diluted by 100 times had no obvious CPE and the anti-CyHV-2 IgY group diluted by 200 times had a small number of cell holes appeared in GiCB cells.However,GiCB cells in the blank control group with CyHV-2 virus solution and the negative control group with ordinary egg powder appeared obvious CPE.The results of the prevention experiment showed that immune protection of that only infected with the virus group,the normal egg powder group(negative control)and the anti-CyHV-2 IgY addition amount was 0.1%,0.3%,and 0.6%of the feed weight of the treatment group is 4.8±8.2%(p>0.05),51.8 ± 10%(p<0.001),57.9 ± 8.4%(p<0.001)and 75.8± 9.5%(p<0.001)compared with the non-challenged normal group.The treatment group with an antibody addition amount of 0.6%has the best the prevention effect among which.The CyHV-2 load in the kidney of the allogynogenetic crucian carp artificially infected with the virus gradually decreased with the increase of anti-CyHV-2 IgY(p<0.05),and the CyHV-21oad in the kidney of the allogynogenetic crucian carp on the verge of death CyHV-2 load is higher than that of the surviving allogynogenetic crucian carp(p<0.05);This result is consistent with the results of the number of CyHV-2 virus particles in each treatment group observed under the transmission electron microscope.Compared with normal fish,the spleen and body and kidney of each experimental group showed different degrees of damage after challenge.Among them,the spleen and body and kidney of fish with anti-CyHV-2 IgY added at 0.6%had less pathological damage that is similar with normal fish group.The results of treatment experiments showed that the immune protection of the negative control group(mixed with ordinary egg powder)and anti-CyHV-2 IgY group were 7.7 ± 7.7%(p>0.05)and 80± 11.5%(p<0.001),respectively.The results of qPCR detection revealed that the CyHV-2 load in the kidneys of the moribund fish and live fish in the blank control group(only fed commercial feed)and the negative control group were not different,while the anti-CyHV-2 IgY group was significantly lower than the blank control groupand negative control group(p<0.05).Among all treatment groups,the live fish in the anti-CyHV-2 IgY group had the lowest viral load(p<0.05).The histopathological section results showed that the spleen and kidney cells in the anti-CyHV-2 IgY group were intact,while the spleen and kidneys in the negative control group and the blank control group were severely damaged after CyHV-2 infection,with tissue necrosis and vacuolization and other pathological phenomena.Serum biochemical index test results showed that compared with normal fish,the activity of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and alkaline phosphatase(ALP)related to fish liver function increased significantly(p<0.05)in the blank control group and the negative control group,while the AST,ALT and ALP activities of the egg yolk antibody group did not increase significantly(p>0.05).The albumin(ALB)content of the blank control group,the negative control group and the egg yolk antibody group all increased significantly(p<0.05),the increase of ALB in the yolk antibody group was significantly lower than that of the blank control group and the negative control group(p<0.05),indicating that the fish’s liver of the yolk antibody group was less damaged.Compared with normal fish,the red blood cell count,hematocrit,hemoglobin content,average hemoglobin concentration,average platelet volume,number and percentage of neutrophils in the blank control group and the negative control group decreased significantly after challenge(p<0.05).The percentage of basophils,the number of basophils,the number of white blood cells and the number of lymphocytes increased significantly(p<0.05);the platelet count and the number of monocytes did not change significantly(p>0.05).In the egg yolk antibody group,compared with the normal fish,the hematocrit,average hemoglobin concentration,neutrophil count and percentage decreased significantly(p<0.05),but the decline was significantly smaller than that of the blank and negative control groups(p<0.05).The percentage of basophils,the number of basophils,and the percentage of lymphocytes increased significantly(p<0.05),but the increase was significantly lower than that of the blank and negative control group(p<0.05).There were no significant changes in other blood routine indicators.Significant change(p>0.05),which indicates that the allogynogenetic crucian carp in the yolk antibody group is infected with CyHV-2,the bleeding degree is small,and the weak anti-infection immune response is triggered.The qPCR analysis showed that the expression levels of interleukin 11(IL-11),IL-6,and complement C3(the third complement component,C3),purine nucleoside phosphorylase(purine nucleoside phosphorylase 5a,Pnp5a),type I interferon(interferon c,IFNc)and lectin(intelectin,ITLN)were up-regulated,while the up-regulation of the expression levels of the egg.yolk antibody group was not significant(p>0.05),Which indicating that the egg yolk antibody can resist CyHV-2 infection and improve the immunity of the fish.After 21 days of challenge,the immune-related gene changes of allogynogenetic crucian carp survived in each treatment group were inconsistent.Compared with the normal group,the relative expression of IL-11 and Pnp5a were all significantly up-regulated(p<0.05)in the blank control group,negative control group and egg yolk antibody group,in which the yolk antibody group had the highest up-regulation fold.Compared with the normal fish,the relative expression of IFNc was up-regulated 1.3 times(p>0.05)and 8.6 times(p<0.05)in the blank control group and the yolk antibody group.The negative control group was down-regulated by 16.5 times(p<0.05).The relative expression of IL-6 of allogynogenetic crucian carp surviving was significantly down-regulated in the blank control group,negative control group and egg yolk antibody group(p<0.05),and the egg-yolk antibody group had the lowest down-regulation factor(p<0.05).There was no difference in the relative expression of C3 between the negative control group and the normal fish(p>0.05),and the egg yolk antibody group was up-regulated by 21.7 times(p<0.05).The relative expression of ITLN was significantly down-regulated(p<0.05)in the three groups,but there was no significant difference(p>0.05).Conclusions:This study successfully induced D4ORFs recombinant protein,prepared anti-CyHV-2 IgY,and determined that freeze-drying is the best anti-CyHV-2 IgY processing method.The results of in vitro antibody neutralization experiments proved that anti-CyHV-2 can reduce the degree of infection of GiCB cells by CyHV-2.The addition of 0.6%of anti-CyHV-2 IgY can effectively prevent the occurrence of gill hemorrhage of allogynogenetic crucian carp;the experimental results of artificial infection of the virus show that oral administration of 0.6%of anti-CyHV-2 IgY feed can significantly increase the infection of CyHV-2 survival rate of allogynogenetic crucian carp.The results of CyHV-2 load showed that treatment with anti-CyHV-2 egg yolk antibody results in reduction in the number of CyHV-2 particles entering the fish;the relative expression levels of immune-related genes,blood and serum indicators,and HE staining of pathological damage tissue all confirmed that anti-CyHV-2 yolk antibody can play a protective role by reducing tissue damage and immune response in fish. |
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