Research On Main Biological Characteristics And Live Attenuated Vaccine Of Yersinia Ruckeri Recovered From Rainbow Trout (Oncorhynchus Mykiss)

Yersinia ruckeri,etiological agent of enteric redmouth diseases(ERM),mainly infects salmonids inducing some clinical signs such as hemorrhages and enteritis.In recent years,ERM outbreaks in farming rainbow trout(Oncorhynchus mykiss)worldwide,causing great economic losses and limiting the development of rainbow trout aquaculture in China.Due to the multi-drug resistance of pathogens and food safety problems,drug usage to control this disease are facing increasingly severe challenges.Therefore,it is of great significance to develop new-type vaccines to prevent the occurrence of ERM.A wide variety of fish species have been described to be susceptible to Y.ruckeri,including Acipenser baerii,Pelteobagrus fulvidraco,Hypophthalmichthys molitrix etc.;while ERM also exhibits a broad geographical distribution.However,little is known about Y.ruckeri recovered from rainbow trout.The virulence of this pathogen is influenced by several factors,such as biofilm,temperature,iron concentration,etc.Furthermore,the secreted toxins like hemolysin are one of the key virulence factors.Herein,in order to provide theoretical basis and guidance for prevent and control of ERM,the experiments were carried out as following:(1)to explore biological characteristics of YR01 isolate;(2)to construct q PCR detection method for clinical diagnosis;(3)to establish the animal infection model;(4)to analyze the transcriptome of rainbow trout spleen after bacterial infection;(5)to verify the function of yhl gene and construct YR01-?yhl B strain;(6)to evaluate the feasibility of YR01-?yhl B as a live attenuated vaccine.The diseased rainbow trout,sampled from a cold-water fish farming located in Qinghai province,exhibited clinical signs such as abnormal feeding and swimming behavior,darken body,exophthalmia,hemorrhages around the mouth and the base of the fins.At necropsy the fish showed bloody ascites,enlarged liver and spleen,and severe enteritis.A dominant strain named YR01 was obtained from liver and spleen of diseased fish under sterile conditions.The artificial infection experiments by i.p.injection indicated that YR01 strain was high pathogenic to rainbow trout juvenile,with a high mortality of 90%.The diseased fish showed the similar clinical signs with those naturally diseased fish.The lethal dose 50(LD₅₀)of YR01 to rainbow trout was estimated to be about 6.5×10⁷ CFU/m L.Histopathological observation showed that the liver,spleen and intestine were damaged to some extent post YR01 infection.YR01 strain was Gram negative and could grow on TSA plate to form 2 mm in diameter,smooth,slightly raised and sticky,milky white colonies with entire edges.Transmission electronic microscope(TEM)observation indicated that each cell contained 6-8 peritrichous flagella anchoring firmly at the cell body.The bacterial cell was about 0.7?m in diameter,1.8-2.7?m in length and 0.14?m in thickness.Combined the Biolog system and 16S r RNA gene sequence analysis,YR01 was identified to be Y.ruckeri.In order to establish a rapid and quantitative method for detection of Y.ruckeri,a q PCR method was constructed through designing of a pair of specific primers YRI-F/R based on rup A gene.The results showed that the primers had good specificity to Y.ruckeri and could be used to effectively amplify target gene.The detection limits were 60 copy/?L by q PCR,about 100 folds lower than conventional PCR.The established q PCR method could be applied in detecting the samples and calculating the pathogen loads in practice.In addition,we constructed a pathological model of ERM disease in rainbow trout by i.p.injection 100 u L Y.ruckeri at a concentration of 2.0×10⁷ CFU/m L to rainbow trout juvenile with the body weight of about 12 g.The pathological model induced obvious clinical signs in rainbow trout with moderate development of ERM,which is more suitable for the later research.To explore the interaction mechanism between rainbow trout and Y.ruckeri,the transcriptomic profiling analysis of spleen samples from rainbow trout at 24 h post-Y.ruckeri infection via RNA-seq was conducted.A total of 147 927 000 clean reads were obtained and 84.81-85.99%data were mapped to the O.mykiss reference genome perfectly.2498 DEGs were identified by comparing these groups,of which2083(83.39%)were up-regulated and 415(16.61%)were down-regulated,in YR-infected fish compared to control fish.GO annotation was next performed by categorizing these DEGs into 23 biological processes(BPs),19 cellular components(CCs),and 16 molecular functions(MFs).KEGG pathway enrichment analyses classified DEGs into many signaling pathways such as NLR,TLR,RLR and cytokine-cytokine receptor interaction signaling.78 immune-related DEGs were further screened,which were mainly distributed in 20 KEGG pathways.The top 3 pathways enriched in these genes included the NOD-like receptor signaling(31 genes),RIG-I-like signaling(35 genes),and Toll-like receptor signaling(51 genes)pathways.Eight DEGs including IL-1?,IL-8,TNF,NOD1,HSP90,CCR9,CARD9 and TLR8a2 were randomly selected to perform q PCR validation,resulting the same expression trend with RNA-Seq and confirming the reliability of transcriptome analysis.The yhl gene of Y.ruckeri was divided into four fragments through protein domain analysis and then prokaryotic expressed to assess the virulence of the products,aiming to uncover the pathogenicity mechanism of this pathogen.The results showed that all four products could affect the proliferation of RTG-2 cells,with the inhibiting rate of 30.77%,26.92%,15.38%and 53.84%,respectively.Expression of IL-2 and IL-1?was up-regulated in RTG-2 cells induced by yhl-B at 72 h and the inflammation occurred.No significant pathological changes were observed in RTG-2 cells inoculated with yhl-A1/A2/A3.RTG-2 cells inoculated with yhl-B were observed to be rounded,exfoliative and form plaques with botryoidal aggregation.The attenuated YR01-?yhl B was constructed by deleting yhl-B gene using G-DOC and?Red recombinant system.There were no significant differences of colonies morphology,biochemical characteristics and growth trait between YR01-?yhl B and wildtype YR01,while antimicrobial susceptibility changed after deletion of yhl B gene.YR01-?yhl B showed lower susceptibility to enrofloxin,cefoperazone,streptomycin and erythromycin,higher susceptibility to kanamycin,amikacin and nitrofurantoin when compared to wild type strain.The cytotoxicity of YR01-?yhl B strain was lower than that of YR01 strain,but the supernatant of the two strains could induce up-regulated expression of IL-?.In vivo experiments showed that both YR01-?yhl B and wild type YR01 were virulent to juveniles of rainbow trout,inducing the up-regulation of immune-related genes in spleen of experimental fish,while the virulence of YR01 was much higher than YR01-?yhl B.Moreover,the feasibility of YR01-?yhl B as a live attenuated vaccine was evaluated.The rainbow trout juvenile was vaccinated by YR01-?yhl B through immersion.The challenge test showed that YR01-?yhl B could protect the experimental fish against Y.ruckeri infection to some degree with an RPS of 66.7%.In this study,the biological characteristics of Y.ruckeri YR01 isolate were investigated and a rapidqPCR detection method and animal infection model were constructed.A batch of DEGs were identified to be involved in immune response of rainbow trout against Y.ruckeri infection.YR01-?yhl B strain was constructed and verified to be able to induce good immune response in rainbow trout,protecting efficiently against Y.ruckeri challenge.The results are of great theoretical and practical significance to develop prevention techniques against ERM.