| Large yellow croaker(Larimichthys crocea)is an economically important marine fish in China.The cultivation of L.crocea has been expanding consistently,yet the deteriorating environment has led to frequent disease outbreaks.The visceral white nodules disease(VWND)caused by Pseudomonas plecoglossicida is one of the serious bacterial diseases,which significantly impairs the healthy development of China’s industry.Vaccination is an effective and environmentally friendly measure for controlling fish diseases caused by different pathogens.Therefore,it is urgent to develop a vaccine that can counteract VWND in large yellow croaker.Live attenuated vaccines(LAVs)can mimic natural infection and evoke strong cell-mediated immune responses and are thus more effective in eliminating pathogenic bacteria than nonreplicating vaccines Several licensed fish vaccines have significantly contributed to the rapid and sustainable development of the aquaculture industry.Currently,no effective commercial vaccine has been developed for the prevention and control of VWND in fish.An immersion challenge model that mimics natural exposure to P.plecoglossicida infection was established.Based on the previously constructed mutant strains,includingΔOmpR,ΔrpoS,and ΔOmpRΔrpoS,we conducted an analysis of the growth curve,motility,biofilm formation,and virulence for each strain.Among them,we selectedΔOmpRΔrpoS for further investigation,specifically focusing on its immunoprotective properties.Then,the use of the mutant ΔOmpRΔrpoS strain as a potential LAV was evaluated,we intraperitoneally immunized L.crocea,measured specific antibody levels in their serum,observed changes in the expression of immune-related genes,recorded bacterial loads in target tissues after challenge,and evaluated relative percent survival(RPS).The results of this study are as follows:1、For the challenge experiment,an immersion challenge model that mimics natural exposure to P.plecoglossicida infection was established.Briefly,the fish were equally divided into 5 groups,and the specimens of 5 groups were immersed in seawater containing different concentrations of 1 × 107 CFU/mL,1 × 106 CFU/mL,1 ×105 CFU/mL,1 × 104 CFU/mL or 1 × 103 CFU/mL wild-type(WT)P.plecoglossicida strain for 30 min,and then transferred into a 1 000 L tank.These fish were observed for clinical signs and mortality for 14 days.To gain a cumulative mortality of approximately 80.00%throughout the challenge experimental period,the immersion dose of 5 × 104 CFU/mL was finally chosen2、The growth curve,motility,biofilm formation,and virulence of the constructed P.plecoglossicida mutant strains ΔOmpR,ΔrpoS,and ΔOmpRΔrpoS were analyzed.We found no significant difference in the in vitro growth kinetics between the mutant and WT strains(P>0.05);the motility of all three mutant strains was significantly lower than that of the WT strain(P<0.01);the biofilm formation capacity ofΔOmpRΔrpoS and ΔrpoS mutant strains was considerably lower than that of the WT strain(P<0.05),while there was no significant difference in biofilm formation capacity between ΔOmpR mutant strain and the WT strain(P>0.05).For large yellow croaker with an average body weight of 40.5 ± 3.5 g,the LD50 of the WT strain PQLYC4 was 1.35 × 101 CFU/fish at 18 ± 2℃ over a 14-day period.In contrast,the LD50 of theΔOmpR and ΔrpoS mutants reached 4.68 × 103 CFU/fish and 2.51 × 105 CFU/fish,respectively,while the LD50 of the ΔOmpRΔrpoS mutant increased to 1.58 × 108 CFU/fish.Since an ideal LAV should be avirulent,the ΔOmpRΔrpoS mutant,which had a 1.17 × 107-fold increase in LD50 value over the parental WT strain,was selected for the vaccination trial.The fish in the PBS control group did not show any typical symptoms,and no mortality occurred.3、In reference to LD50 of the ΔOmpRΔrpoS mutant strain for L.crocea,fish were immunized by intraperitoneally injection of 200 μL ΔOmpRΔrpoS P.plecoglossicida solution(1 × 107 CFU/mL)per fish.Fish in the mock-vaccinated control group were injected with 200 μL of PBS per fish.Booster immunizations were performed in the same way 14 days after the initial vaccination.The serum of the vaccinated and control fish was collected at 1,2,3,4,5,6,7,and 8 wpiv and analyzed by ELISA.At 4 wpiv and 6 wpiv,the large yellow croakers were challenged separately by immersion using the WT P.plecoglossicida strain,the RPS was calculated.Spleen and head kidney tissues from 5 fish of each group were collected at 24 h post-challenge for the expression analysis of immune genes by quantitative real-time PCR(qPCR)and spleen tissues were collected from 5 fish in each group at 5 d post-challenge to quantify the bacterial load.The results showed that the P.plecoglossicida specific IgM antibody level in serum increased significantly(P<0.05)at 3 wpiv and peaked at 5 wpiv.There was a subsequent decline but remained significantly(P<0.05)higher than the control group at 6,7,and 8 wpiv.the immune-related genes(IgM,IgT,MHCIIα,CD4-1,MHCI,CD8α,and ZAP70)were remarkably upregulated after P.plecoglossicida challenge,and a lower bacterial load was observed in the spleen of these fish,unlike the control fish,which had high bacterial loads.Fish vaccinated twice via intraperitoneal injection with the ΔOmpRΔrpoS strain had a RPS of 72.25%and 64.90%,when challenged at 4 and 6 wpiv,respectively.In addition,evaluate the impact of the ΔOmpRΔrpoS mutant on fish growth.During the 8-week experimental period,had no mortality and did not exhibit any pathological changes in internal organs,including spleen and kidney,similar to observations made in fish injected with 200 μL PBS throughout the experimental period.Fish were weighed at 2-weeks intervals over a period of 8 weeks to investigate the impact of the ΔOmpRΔrpoS mutant on their growth.Taken together,these results suggest that the ΔOmpRΔrpoS mutant is a promising LAV candidate against P.plecoglossicida infection. |
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