| Aeromonas salmonicida is a pathogen known to cause furunculosis in Salmonidae,resulting in serious economic losses to the salmonid fish farming industry.In the past few decades,though there have been many studies on the prevention of furunculosis,it remains prevalent in many fisheries.Therefore,there is an urgent need to develop a cost-effective vaccine for preventing A.salmonicida infection.The emergence of many new subtypes and the expansion of the host range each year poses a huge threat to the cultivation of a variety of marine and freshwater fishes worldwide.At present,Although there was some reports of infection by atypical strains on the C.auratus,the pathogenesis and tissue pathology remain unclear..This study was undertaken to clarify the biological characteristics of a newly isolated atypical A.salmonicida strain and the immune response mechanism of C.auratus to infection with this pathogen,and to investigate candidate vaccines against typical A.salmonicida in salmonids.Whole-genome sequencing technology,transcriptomics,histopathology,recombinant adenovirus,and other molecular biology technologies were used in this study the following results were obtained:(1)A second-generation whole-genome sequencing technology was used to sequence the genome of a strain of A.salmonicida isolated from C.auratus.Analysis of the genome components and gene function annotation revealed that the average GC content of the strain was 58.6%,the total genome length 4,656,393 bp,and the number of coding genes was 4,226.A total of 109 non-coding RNAs were found,of which 102 were tRNAs and 7 were sRNAs,accounting for 0.1723% and 0.0227% of the total genome,respectively.A prophage with a total length of 29,875 bp and 8 gene islands with a total length of 97,861 bp were also found.Secreted proteins were predicted to recognize 374 proteins with a signal peptide structure and 1,032 proteins with a transmembrane structure.Secretory system proteins were predicted to recognize 13 effector proteins of the T2 SS type secretion system and 5 of the T6 SS type secretion system and 127 of the T3 SS type secretion system.A total of 424 virulence factors were found,which included hemolysin,O antigen,flagellin,S-layer protein,OmpA,T3 SS,extracellular polysaccharide,capsular polysaccharide,and other pathogenic virulence factors.Thirty genes related to drug resistance were also identified,and these are mainly resistant to aminoglycosides,fluoroquinolones,tetracycline,macrolides,and chloramphenicol inter alia.These results provide a theoretical basis for further functional genomics research and development of new prevention and treatment protocols.(2)Transcriptomics and histopathology were used to analyze the immune response and lesions of C.auratus infected with A.salmonicida.Comparative analysis showed that differentially expressed genes(DEGs)(3428 downregulated and 3151 upregulated)were identified on day 5 post-infection(5 dpi).Further annotation and analysis revealed that the DEGs were enriched in enzyme regulator activity,response to oxidative stress,iron ion homeostasis and other functions,and mitogen-activated protein kinase(MAPK),factor-κB(NF-κB),toll-like receptor(TLR),and nucleotide-binding oligomerization domain(NOD)-like receptor(NLR)etc.,and immune-related signaling pathways.The four C-type lysozyme genes found in all DEGs were significantly upregulated after infection.Furthermore,there was severe bleeding in the body of the infected fish.In addition to this,the intestine,liver,spleen,and kidney showed varying degrees of inflammatory damage,particularly goblet cell hyperplasia of intestinal mucosa epithelium and degeneration and necrosis of renal tubular epithelial cells.Concurrently,with the increase in A.salmonicida concentration,the cumulative mortality increased and the severity of lesions in the hindgut and head-kidney tissues increased.The relative expression levels of four immune-related genes(TNF-α,IL-1β,IL-11,and C-lysozyme)were upregulated significantly compared with those in the control group(P<0.05).Therefore,the regulation of these genes and signaling pathways plays an important role in maintaining the stability of the internal environment and resisting infection by pathogenic bacteria.In addition,the pathological characteristics of the infected crucian carp are different from other fish.In summary,this study provides a scientific basis for the immune response mechanism and histopathological diagnosis of C.auratus resistant to atypical A.salmonicida infection.(3)Based on the results of the previous step,this study used qRT-PCR,Western blotting,prokaryotic expression,in vitro bacteriostatic experiments,and other methods to further analyze the expression characteristics and antibacterial characteristics of C-type lysozyme after C.auratus was infected.The results showed that the gene and protein expression of C-type lysozyme in the liver,spleen,kidney,and hindgut of infected C.auratus were significantly upregulated.The gene was most significantly upregulated in the liver,15 times more than uninfected.In addition,the recombinant lysozyme showed obvious antibacterial activity against A.salmonicida.With an increase in the dosage of the recombinant lysozyme,the radius of the bacteriostatic circle seen on the culture dish became larger.When 40 μg of recombinant lysozyme was used,the average radius of the bacteriostatic circle reached 0.92 cm.Therefore,the C-type lysozyme of crucian carp plays an important role in resisting the infection of Aeromonas salmonicida.These studies not only revealed the characteristics of the immune response of C-type lysozyme after the C.auratus were infected with A.salmonicida,but also provided a scientific basis for solving drug resistance in the future.(4)Using recombinant adenovirus technology,a recombinant adenovirus that can express protective antigen A layer protein was successfully constructed.The immune protection rate and specific antibody levels in the peripheral blood were then determined after immunizing rainbow trout.In addition,relative levels of IgM and IgT in the head kidney and hindgut before and after immunization were measured by qRT-PCR.Western blotting results indicated that the recombinant adenovirus could infect HEK-293 cells and express the A layer protein(encoded by Vapa).Further,survival analysis of fish 28 days after challenge showed that immunization significantly lowered the mortality rate(40%)compared to that in the control group(76.6%)and empty vector group(73.6%).This also led to an increase in specific antibodies in the peripheral serum.Additionally,levels of IgM and IgT in the head kidney and hindgut increased to varying degrees,especially the doubling of IgT in the intestine.Therefore,the inoculation of recombinant adenovirus can activate the humoral immune response of rainbow trout,and it has an immunoprotective effect on the infection of Aeromonas salmonicida.In conclusion,our research provides a candidate vaccine for the prevention of A.salmonicida strain A450 infection in rainbow trout and lays the foundation for future research on adaptive immune mechanisms associated with rainbow trout antibodies. |
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