Construction Of Pseudomonas Plecoglossicida Bacterial Ghost And Evaluation Of Immune Efficacy

Large yellow croaker（Larimichthys crocea,LYC）is an important economic marine fish,and the aquaculture output has been the first among maricultured fish in China since2014.With the development of intensive high-density aquaculture mode,many kinds of diseases of LYC have broken out.Visceral granulomas disease（VGD）caused by Pseudomonas plecoglossicida infection has a high incidence and mortality,and has become the main bacterial diseases threatening the healthy development of aquaculture industry of LYC.At present,enrofloxacin or doxycycline hydrochloride is generally used for prevention and control of the disease in production,but the clinical effect is not stable,and it is easy to induce drug-resistant strains,causing potential threats to the prevention and control of the disease.Therefore,it is urgent to develop a green and efficient control method.As a biological control method,vaccine has been widely used to control aquaculture disease,with the advantages of safety and efficiency.However,the reported vaccines against VGD of LYC so far all have the shortcomings of low efficiency or poor safety（e.g.live attenuated vaccine）.Therefore,to design and screen a safer and more effective vaccine candidate is of great significance to the development of the LYC industry.Bacterial ghosts are inactivated gram-negative bacteria with empty and intact bacterial envelopes,which are produced by controlled expression of phage Phi X174 lysis gene E and devoid of cytoplasmic content.The ghosts retain almost the same surface structures（including antigen determinants,immune adhesion factors etc.）as the original live bacterial,thus can induce both humoral and cellular immune responses.Therefore,it has the potential to be developed as a candidate vaccine,ideal vaccine adjuvant or even vaccine carrier.In this study,E gene and kanamycin resistance gene were introduced into the commercial plasmid p BV220 to construct a new temperature-controlled-lysis plasmid named 220E-Kn through genetic engineering.The recombinant plasmid 220E-Kn was then introduced into Escherichia coli or P.plecoglossicida isolated from VGD carried LYC to evaluate its ability to induce ghosts formation.The results showed that the plasmid220E-Kn could be stably passaged in E.coli and start the high-efficiency expression of lysis gene at 42°C.After 4 hours induction,the lysis efficiency reached 99.997%.The SEM results showed the E.coli experienced structural changes such as lysis,cell contraction and content leakage.Fllowed,the plasmid 220E-Kn was introduced into P.plecoglossicida by electric shock and selected by kanamycin to obtain positive transformants.After three generations culture at 28°C,the plasmid 220E-Kn was proved to be stably passaged in P.plecoglossicida under the kanamycin pressure.The results of lysis induction showed that the lysis efficiency of transformant reached 100%afer 24 h induction at 42°C.The SEM results showed that the ghosts had the same features as the E.coli ghosts.After 24 h induction,the bacteria solution was resuscated and cultured on TSA for 48 h,and no colony formation unit was found,indicating complete lysis and no active bacteria remained.In order to accurately evaluate the immune protective effect of P.plecoglossicida ghosts,a preliminary study was conducted on the model of P.plecoglossicida infection on LYC.Under the water temperature 20°C to 25°C,the healthy LYC（35±5 g）was infected with P.plecoglossicida either by intraperitoneal injection or immersion.The results showed that both infection methods could cause effective infection and death,while the death trend of immersion was much closer to the natural infection,with typical VGD formulated in the spleen.Besides,a certain proportion of operational injuries related deaths were observed in IP injection group,and the repeatability among parallel groups was poor.Therefore,immersion infection model was recommended through our study.To evaluate the virulence of P.plecoglossicida under low water temperature（10°C～13°C）,an immersion infection trial was carried out.The results showed that the LD₅₀ of P.plecoglossicida to LYC（35±5 g）infected by immersion under temperature 10°C～13°C was 1.3×10⁴ CFU/m L.Based on all data collected above,we made a challenge by immersion at the dose of 2×10⁴ CFU/m L at 28 days after immunization to evaluate the RPS（Relative percentage survival）of the two vaccines—the ghost vaccine and the formaldehyde inactivated vaccine.The results showed that the RPS of the ghost vaccine was 66.67%at 14 days after challenge,compared to 46.67%of the formaldehyde inactivated vaccine.Oberviously,the ghost vaccine had a stronger protective effect than formaldehyde inactivated vaccine.In order to further evaluate the immune protective effect of ghost vaccine,fish serum,head kidney and spleen of three groups（PBS,formaldehyde killed cells vaccine,FKC and bacterial ghost vaccine,BG）were collected,and the parameters related to innate and acquired immunity were monitored,including serum antibody levels,AKP and ACP levels in serum,the relative transcription levels of immune related genes Ig M,IL-1β,MHCIα,MHCIIβ,CD4 and CD8.The ELISA results showed that the antibody titers in serum of BG group were higher than both the PBS and FKC group during the whole 10-week immunization period,specifically,at the 8th and 10th weeks past vaccination,the BG group showed a significantly higher serum titers than FKC group（P<0.01）,correspondingly,the transcription level of gene Ig M in spleen of BG vaccined at the 6th week was significantly higher than FKC group（P<0.01）.In other side,the AKP and ACP levels in BG group were significantly higher than FCK group at 8th week（P<0.05）.When it turned to cellular immune-related parameters,we found that the transcription levels of IL-1βin the head kidney and spleen of BG group had a same trend of increasing first and then decreasing gradually and both were significantly higher than FKC group at the 4th and 6th weeks past vaccination（P<0.01）.Furthermore,the transcription levels of CD4 and CD8 showed different expression patterns in the head kidney and spleen.They all showed a trend of gradual decline in the head kidney.The transcription levels of CD4 in head kidney of BG group were significantly higher than that of FKC group at the 2th and4th weeks（P<0.01）,while in spleen,no significant changes were detected during the whole immunization period.The transcription levels of CD8 in spleen of BG group showed a trend of increasing first and then decreasing and they were significantly higher than that of the FKC group at 4th week（P<0.05）.Briefly,in this study,we successfully constructed a temperature-controlled-lysis strain of P.plecoglossicida isolated from LYC suffered VGD,this strain,named 220E-kn,when induced at 42°C for 24 hours,cells were completely lysed to form bacterial ghost.The RPS of ghost vaccine was 66.67%,which was higher than that of FKC vaccine（46.67%）.Meanwhile,the monitoring results of immune-related parameters showed that the ghost could induce stronger and more lasting immunity,thus is expected to be developed as a safe and efficient candidate vaccine in future.